A new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120

In silico analysis followed by experimental validation leads us to propose that the predicted protein All0195 of Anabaena sp. PCC7120 showing enhanced expression under sodium arsenate (Na₂HAsO₄) stress belongs to the thioredoxin superfamily with structural similarity to bacterial arsenate reductase. The All0195 protein demonstrated C-X-TC-X-K, NTSG-X₂-YR, and D-X₂-L-X-KRP as functional motifs that show similarity to seven known bacterial arsenate reductase family protein homologs with Cys, Arg, and Pro as conserved residues. In view of physicochemical properties, such as aliphatic index, ratio of Glu?+?Lys to Gln?+?His, and secondary structure, it was evident that All0195 was also a thermostable protein. The predicted three-dimensional structure on molecular docking with arsenate oxyanion ( $ HAsO_4^{- 2 } $ ) revealed its interaction with conserved Cys residue as also known for other bacterial arsenate reductase. In silico derived properties were experimentally attested by cloning and heterologous expression of all0195. Furthermore, this protein functionally complemented the arsenate reductase-deficient sodium arsenate-hypersensitive phenotype of Escherichia coli strainWC3110 (ΔarsC) and depicted arsenate reductase activity on purification. In view of the above properties, All0195 appears to be a new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120.     

Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic

In cyanobacteria, the biosynthesis of the phycobiliprotein and phytochrome chromophore precursor phycocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduction of biliverdin IXalpha. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native M(r) of 30,400 +/- 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IXalpha. The enzyme exhibits a strong preference for plant type [2Fe-2S] ferredoxins; however, flavodoxin can also serve as an electron donor. HPLC analyses establish that catalysis proceeds via the two electron-reduced intermediate 18(1),18(2)-dihydrobiliverdin, indicating that exovinyl reduction precedes A-ring (endovinyl) reduction. Substrate specificity studies indicate that the arrangement of the A- and D-ring substituents alters the positioning of the bilin substrate within the enzyme, profoundly influencing the course of catalysis. Based on these observations and the apparent lack of a metal or small molecule cofactor, a radical mechanism for biliverdin IXalpha reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.     

Identification of amino acid residues essential to the activity of lyase CpcT1 from Nostoc sp. PCC7120

The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1.     

Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120.

The structural gene for ferredoxin I, petF, from the cyanobacterium Anabaena sp. strain PCC 7120 has been isolated from a recombinant lambda library. Mixtures of tetradecanucleotides and heptadecanucleotides, each containing all possible DNA sequences corresponding to two separate regions of the ferredoxin amino acid sequence, were synthesized and used as hybridization probes to identify a genomic clone containing the coding sequence for the petF gene. The sequence of the entire petF coding region and portions of the 3'- and 5'-flanking regions was determined. The DNA sequence of petF suggests that, in contrast to the nucleus-encoded plant protein, cyanobacterial apoferredoxin is not synthesized as a higher-molecular-weight precursor. The Anabaena petF gene is a single-copy gene. During growth on complete medium it was transcribed into a monocistronic mRNA species of approximately 500 bases that initiated 100 base pairs upstream from the petF coding region.     

Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120

Heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 occurs at regular intervals under nitrogen starvation and is regulated by a host of signaling molecules responsive to availability of fixed nitrogen. The heterocyst differentiation inhibitor PatS contains the active pentapeptide RGSGR (PatS-5) at its C-terminus considered the minimum PatS fragment required for normal heterocyst pattern formation. PatS-5 is known to bind HetR, the master regulator of heterocyst differentiation, with a moderate affinity and a submicromolar dissociation constant. Here we characterized the affinity of HetR for several PatS C-terminal fragments by measuring the relative ability of each fragment to knockdown HetR binding to DNA in electrophoretic mobility shift assays and using isothermal titration calorimetry (ITC). HetR bound to PatS-6 (ERGSGR) >30 times tighter (K(d) = 7 nM) than to PatS-5 (K(d) = 227 nM) and >1200 times tighter than to PatS-7 (DERGSGR) (K(d) = 9280 nM). No binding was detected between HetR and PatS-8 (CDERGSGR). Quantitative binding constants obtained from ITC measurements were consistent with qualitative results from the gel shift knockdown assays. CW EPR spectroscopy confirmed that PatS-6 bound to a MTSL spin-labeled HetR L252C mutant at a 10-fold lower concentration compared to PatS-5. Substituting the PatS-6 N-terminal glutamate to aspartate, lysine, or glycine did not alter binding affinity, indicating that neither the charge nor size of the N-terminal residue's side chain played a role in enhanced HetR binding to PatS-6, but rather increased binding affinity resulted from new interactions with the PatS-6 N-terminal residue peptide backbone.     

鱼腥蓝细菌PCC7120铁吸收机制

铁离子是鱼腥蓝细菌PCC7120进行呼吸作用、光合作用和固氮作用中相关酶的重要辅基之一,缺铁将严重影响蓝细菌的生存.富氧的生态环境中铁通常以不溶的Fe3+形式存在,不易被细胞吸收利用.低铁条件下,鱼腥蓝细菌PCC7120分泌能螯合铁离子的嗜铁素,通过外膜上相应的转运体将嗜铁素-铁复合物转运到细胞内.综述了近年来在嗜铁素的种类及其生物合成途径、铁吸收系统的组成和功能等方面的最新进展,分析了铁吸收系统的调控机制,为进一步开展鱼腥蓝细菌铁吸收机制的研究提供依据.     

生物反应器培养转基因鱼腥藻的研究

在反应器中研究了转人TNF-α基因鱼腥藻7120(Anabaena sp．PCC7120,pDC-TNF)的培养。结果表明气升式反应器适合于转基因鱼腥藻的培养。气升式反应器中通气量和光照是主要的影响因素,观察到1L罐中最适通气量为60～75L／h,最适光照强度为1200lx,此时在25℃混养,光照时间／黑暗时间为12h／12h,15d生物量干重大于3g／L,TNF表达水平约占总可溶蛋白的22％,达到了摇瓶培养水平。实验发现添加维生素B1 300μg／L、B12 200μg／L和生物素4μg／L时,生产周期为12d,缩短20％,表达水平相同。培养过程通入含有5％CO2的空气,能促进生长,缩短生产周期,但收获生物量不受影响。从添加维生素和通入CO2的培养结果证明反应器中培养时,光照是限制性因素,当反应器系统一定时,最终生物量有一个最大值,如需进一步提高产量,必须设法改变光照系统。     

Characterization of HetR protein turnover in Anabaena sp. PCC 7120

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied. Received: 24 June 1997 / Accepted: 5 December 1997     

A novel genome rearrangement involved in heterocyst differentiation of the cyanobacterium Anabaena sp. PCC 7120

Abstract A new procedure for the preparation of intact microbial DNA allowed us to obtain DNA, suitable for pulsed-field gel electrophoresis, from both vegetative cells and heterocysts (differentiated cells with a potential for nitrogen fixation) of the cyanobacterium Anabaena PCC 7120. Through this procedure it was possible to locate genomic developmental rearrangements by visualizing the increased mobility of large heterocyst DNA fragments undergoing rearrangements. The 390-kb Sal I fragment of vegetative cell DNA was shown to lose about 70 kb as a result of the previously reported 11- and 55-kb deletions, restoring functional nif operons. A new developmental rearrangement was also detected. This takes place more than 600 kb upstream of the nif operons and results in the excision of about 18 kb from the 505-kb fragment.     

Essential residues for DNA binding activity of ManR fromAnabaena sp. PCC 7120

Cyanobacterial ManR is a member of the OmpR family of response regulator that regulates the expression of themntABC andmntH in response to Mn²⁺ signals. Single-alanine substitutions of I204, L207 and R208 residues of the ManR, which constituted the DNA recognition helix, were obtained by the overlap extension method of PCR. EMSA was used to detect the complexes of the proteins of ManR mutants I204A, L207A, R208A, and the DNA fragment of promoter region of themntH gene fromAnabaena sp. PCC 7120. Results showed the formation of the complexes of the proteins of ManR mutants and DNA could not be detected, indicating that the mutagenesis of the residues I204, L207 and R208 in the ManR HTH domain could lead to the elimination of DNA binding activity of the ManR. Homologous analysis showed that residues I204, L207 and R208 of the ManR are also conservative in the αhelix 3 region of effector domain of other proteins of OmpR/PhoB subfamily, indicating that they are essential residues for DNA binding activity. No significant alteration between wild type and mutant proteins was detected by Far-UV CD spectra at the secondary structure level.     

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml^?1), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.     

FtsZ degradation in the cyanobacterium Anabaena sp. strain PCC 7120

In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Histidine tag pull-downs were used to address this question. However, the main observation was that FtsZ is a target of proteolysis. Experiments using various cell-free extracts, an unrelated protein, and protein blot analyses further supported the idea that FtsZ is proteolytically cleaved in a specific manner. In addition, we show evidence that both FtsZ termini seem to be equally prone to proteolysis. Taken together, our data suggest the presence of an unknown player in cyanobacterial cell division, opening up the possibility to investigate novel mechanisms to control cell division in Anabaena PCC 7120.     

Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

The aim of the present study was to investigate the tolerance of five new Achromobacter and Pseudomonas strains to kerosene and to establish if the production of several secondary metabolites increases or not when these bacteria were grown in the presence of kerosene. The biodegradation of kerosene by isolated bacteria was also investigated in this study. Five Proteobacteria were isolated from different samples polluted with petroleum and petroleum products. Based on their morphological, biochemical, and molecular characteristics, isolated bacteria were identified as Achromobacter spanius IBBPo18 and IBBPo21, Pseudomonas putida IBBPo19, and Pseudomonas aeruginosa IBBPo20 and IBBPo22. All these bacteria were able to tolerate and degrade kerosene. Higher tolerance to kerosene and degradation rates were observed for P. aeruginosa IBBPo20 and IBBPo22, compared with that observed for A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19. All these bacteria were able to produce several secondary metabolites, such as surfactants and pigments. Glycolipid surfactants produced by P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 have a very good emulsification activity, and their activity increased when they were grown in the presence of kerosene. The production of rhamnolipid surfactants by P. aeruginosa IBBPo20 and IBBPo22 was confirmed by detection of rhlAB gene involved in their biosynthesis. Pyocyanin and pyoverdin pigments were produced only by P. aeruginosa IBBPo20 and IBBPo22, while carotenoid pigments were produced by all the isolated bacteria. Significant changes in pigments production were observed when P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 were grown in the presence of kerosene. Due to their ability to tolerate and degrade kerosene, and also to produce several secondary metabolites, the isolated bacteria could be used in the bioremediation of kerosene-polluted environments.     

Anabaena sp. strain PCC 7120 hetY gene influences heterocyst development

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.